Method for producing the FnuDI restriction endonuclease and methylase

ABSTRACT

The present invention is directed to a method for cloning and producing the FnuDI restriction endonuclease by (1) introducing the restriction endonuclease gene from F. nucleatum D into a host whereby the restriction gene is expressed; (2) fermenting the host which contains the vector encoding and expressing the FnuDI restriction endonuclease, and (3) purifying the FnuDI restriction endonuclease from the fermented host which contains the vector encoding and expressing the FnuDI restriction endonuclease activity.

BACKGROUND OF THE INVENTION

The present invention relates to clones for the FnuDI restrictionendonuclease and modification methylase, and to the production of theseenzymes from the clones.

Restriction endonucleases are a class of enzymes that occur naturally inbacteria. When they are purified away from other contaminating bacterialcomponents, restriction endonucleases can be used in the laboratory tobreak DNA molecules into precise fragments. This property enables DNAmolecules to be uniquely identified and to be fractionated into theirconstituent genes. Restriction endonucleases have proved to beindispensable tools in modern genetic research. They are the biochemical'scissors' by means of which genetic engineering and analysis isperformed.

Restriction endonucleases act by recognizing and binding to particularsequences of nucleotides (the 'recognition sequence') along the DNAmolecule. Once bound, they cleave the molecule within, or to one sideof, the sequence. Different restriction endonucleases have affinity fordifferent recognition sequences. Over one hundred different restrictionendonucleases have been identified among many hundreds of bacterialspecies that have been examined to date.

Bacteria usually possess only a small number restriction endonucleasesper species. The endonucleases are named according to the bacteria fromwhich they are derived. Thus, the species Haemophilus aegyptius, forexample synthesizes 3 different restriction endonucleases, named HaeI,HaeII and HaeIII. These enzymes recognize and cleave the sequences(AT)GGCC(AT), PuGCGCPy and GGCC respectively. Escherichia coli RY13, onthe other hand, synthesizes only one enzyme, EcoRI, which recognizes thesequence GAATTC.

While not wishing to be bound by theory, it is thought that in nature,restriction endonucleases play a protective role in the welfare of thebacterial cell. They enable bacteria to resist infection by foreign DNAmolecules like viruses and plasmids that would otherwise destroy orparasitize them. They impart resistance by binding to infecting DNAmolecule and cleaving them each time that the recognition sequenceoccurs. The disintegration that results inactivates many of theinfecting genes and renders the DNA susceptible to further degradationby exonucleases.

A second component of bacterial protective systems are the modificationmethylases. These enzymes are complementary to restriction endonucleasesand they provide the means by which bacteria are able to protect theirown DNA and distinguish it from foreign, infecting DNA. Modificationmethylases recognize and bind to the same nucleotide recognitionsequence as the corresponding restriction endonuclease, but instead ofbreaking the DNA, they chemically modify one or other of the nucleotideswithin the sequence by the addition of a methyl group. Followingmethylation, the recognition sequence is no longer bound or cleaved bythe restriction endonuclease. The DNA of a bacterial cell is alwaysfully modified, by virtue of the activity of its modification methylaseand it is therefore completely insensitive to the presence of theendogenous restriction endonuclease. It is only unmodified, andtherefore identifiably foreign, DNA that is sensitive to restrictionendonuclease recognition and attack.

With the advent of genetic engineering technology, it is now possible toclone genes and to produce the proteins and enzymes that they encode ingreater quantities than are obtainable by conventional purificationtechniques. The key to isolating clones of restriction endonucleasegenes is to develop a simple and reliable method to identify such cloneswithin complex 'libraries', i.e. populations of clones derived by'shotgun' procedures, when they occur at frequencies as low as 10⁻³ to10⁻⁴. Preferably, the method should be selective, such that theunwanted, majority, of clones are destroyed while the desirable, rare,clones survive.

Type II restriction-modification systems are being cloned withincreasing frequency. The first cloned systems used bacteriophageinfection as a means of identifying or selecting restrictionendonuclease clones (HhaII: Mann et al , Gene 3: 97-112, (1978); EcoRII:Kosykh et al., Molec. gen. Genet 178: 717-719, (1980); PstI: Walder etal., Proc. Nat. Acad. Sci. USA 78 1503-1507, (1981)). Since the presenceof restriction-modification systems in bacteria enables them to resistinfection by bacteriophages, cells that carry clonedrestriction-modification genes can, in principle, be selectivelyisolated as survivors from libraries that have been exposed to phage.This method has been found, however, to have only limited value.Specifically, it has been found that cloned restriction-modificationgenes do not always manifest sufficient phage resistance to conferselective survival.

Another cloning approach involves transferring systems initiallycharacterized as plasmid-borne into E. coli cloning plasmids (EcoRV:Bougueleret et al., Nucleic Acids Res. 12:3659-3676, (1984); PaeR7:Gingeras and Brooks, Proc. Natl. Acad. Sci. USA 80:402-406, (1983);Theriault and Roy, Gene 19:355-359, (1982); PvuII: Blumenthal et al.,J.BacterioI. 164:501-509, (1985)).

A third approach, and one that is being used to clone a growing numberof systems, involves selecting for an active methylase gene referring toour patent application No. 707079 and (BsuRI: Kiss et al., Nucleic AcidsRes. 13:6403-6421, (1985)). Since restriction and modification genestend to be closely linked, clones containing both genes can often beisolated by selecting for just the one gene. Selection for methylationactivity does not always yield a complete restriction-modificationsystem however, but instead sometimes yields only the methylase gene(BspRI: Szomolanyi et al., Gene 10:219-225, (1980); BcnI: Janulaitis etal, Gene 20: 197-204 (1982); BsuRI: Kiss and Baldauf, Gene 21: 111-119,(1983); and MspI: Walder et al., J Biol. Chem. 258:1235-1241, (1983)).

A potential obstacle to cloning restriction-modification genes lies intrying to introduce the endonuclease gene into a host not alreadyprotected by modification. If the methylase gene and endonuclease geneare introduced together as a single clone, the methylase mustprotectively modify the host DNA before the endonuclease has theopportunity to cleave it. On occasion, therefore, it might only bepossible to clone the genes sequentially, methylase first thenendonuclease. Another obstacle to cloning restriction-modificationsystems lies in the discovery that some strains of E.coli reactadversely to cytosine modification; they possess systems that destroyDNA containing methylated cytosine (Raleigh and Wilson, Proc. Natl.Acad. Sci., USA 83:9070-9074, (1986)). Cytosine-specific methylase genescannot be cloned easily into these strains, either on their own, ortogether with their corresponding endonuclease genes. To avoid thisproblem it is necessary to use mutant strains of E.coli (McrA⁻ andMcrB⁻) in which these systems are defective.

Because purified restriction endonucleases, and to a lesser extent,modification methylases, are useful tools for characterizing andrearranging DNA in the laboratory, there is a commercial incentive toobtain strains of bacteria through recombinant DNA techniques thatsynthesize these enzymes in abundance. Such strains would be usefulbecause they would simplify the task of purification as well asproviding the means for production in commercially useful amounts.

SUMMARY OF THE INVENTION

In accordance with the present invention there is provided a clonecontaining the genes for the FnuDI restriction endonuclease andmodification methylase derived from Fusobacterium nucleatum D, as wellas related methods for the production of the enzymes. More specifically,this invention relates to clones which express the restrictionendonuclease FnuDI, an enzyme which recognizes the DNA sequence GGCC andcleaves between the G and C. See Lui, A. C. P., McBride, B. C., Vovis,G. F. and Smith, M., Nucleic Acids Res. 6:1-15, (1979), the disclosureof which is hereby incorporated by reference herein. FnuDI restrictionendonuclease produced in accordance with the present invention is freeof the contaminanting FnuDII and FnuDIII endonucleases present inF.nucleatum D.

The preferred method for cloning this enzyme comprises forming a librarycontaining the DNA from F.nucleatum D, isolating those clones whichcontain DNA coding for the FnuDI modification methylase and screeningamong these to identify those that also contain the FnuDI restrictionendonuclease gene.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the scheme for cloning and producing the FnuDIrestriction endonuclease.

FIG. 2 is a restriction map of a 5.5 Kb HindIII fragment of F.nucleatumD DNA that encodes the FnuDI restriction endonuclease and modificationmethylase. The fragment was cloned into the HindIII site of pBR322 (ATCC37017) to create pFnuDIRM 2-33, then it was transferred into the HindIIIsite of pUC19 (ATCC 37254) to create pFnuDIRM 102-1.

FIG. 3 is a photograph of an agarose gel demonstrating FnuDI restrictionendonuclease activity in a cell extract of E.coli RR1 (ATCC 31343)carrying pFnuDIRM 2-33.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to clones of the FnuDI restriction andmodification genes, as well to the restriction endonuclease FnuDIproduced from such clones. The FnuDI genes are cloned by a method whichtakes advantage of the fact that certain clones which are selected onthe basis of containing and expressing the FnuDI modification methylasegene also contain the FnuDI restriction gene. The DNA of such clones isresistant to digestion by the FnuDI restriction endonuclease and theHaeIII restriction endonuclease. The resistance to digestion affords ameans for selectively isolating clones encoding the FnuDI methylase andrestriction endonuclease.

The method described herein by which the FnuDI restriction gene andmethylase gene are preferably cloned and expressed are illustrated inFIG. 1, and they include the following steps:

1. The DNA of Fusobacterium nucleatum D is purified. F.nucleatum D hasbeen described by Lui et al, supra, and was provided to us by Dr.Michael Smith, Dept. of Biochemistry, Faculty of Medicine, TheUniversity of British Columbia, Vancouver, Canada.

2. The DNA is digested with a restriction endonuclease such as HindIII.

3. The digested DNA is ligated to a cloning vector such as pBR322 (ATCC37017), that contains one or more FnuDI sites. The ligated DNA istransformed into an appropriate host such as Escherichia coli strain RR1(ATCC 31343).

4. The transformed mixture is plated onto media selective fortransformed cells, such as the antibiotic ampicillin. After incubation,the transformed colonies are collected together into a single culture,the cell library.

5. The recombinant plasmids are purified in toto from the cell libraryto make the plasmid library.

6. The plasmid library is digested to completion with the FnuDIrestriction endonuclease, or with an equivalent endonuclease, such asHaeIII, from Haemophilus haemolyticus. FnuDI (or HaeIII) digestiondifferentially destroys unmodified, non-methylase-containing, clones,increasing the relative frequency of FnuDI methylase clones.

7. The digested plasmid library is transformed back into an appropriatehost such as E.coli RR1 and transformants are recovered by plating ontoselective media. The colonies are picked and their DNA is analyzed forthe presence of the FnuDI modification gene: the plasmids that theycarry are purified and incubated with the FnuDI (or HaeIII) restrictionendonuclease to determine whether they are resistant to digestion. Totalcellular DNA (chromosomal and plasmid) is also purified and incubatedwith the FnuDI (or HaeIII) restriction endonuclease. The DNA of clonesthat carry the FnuDI modification gene should be fully modified, andboth plasmid DNA and total DNA should be substantially resistant todigestion.

8. Clones carrying the FnuDI restriction endonuclease are identified bypreparing cell extracts of the FnuDI methylase clones, identified instep 8, and assaying the extracts for FnuDI restriction endonucleaseactivity.

9. The quantity of FnuDI restriction endonuclease produced by the clonesmay be increased by elevating the gene dosage, through the use of highcopy number vectors, and by elevating the transcription rate, throughthe use of highly active, exogenous promotors.

10. The FnuDI restriction endonuclease may be produced from clonescarrying the FnuDI restriction and modification genes by propagation ina fermenter in a rich medium containing ampicillin. The cells arecollected by centrifugation and disrupted by sonication to produce acrude cell extract containing the FnuDI restriction endonucleaseactivity.

11. The crude cell extract containing the FnuDI restriction endonucleaseactivity is purified by standard protein purification techniques such asaffinitychromatography an ion-exchange chromatography.

Although the above-outlined steps represent the preferred mode forpracticing the present invention, it will be apparent to those skilledin the art that the above described approach can vary in accordance withtechniques known in the art. The following example is given toillustrate embodiments of the present invention as it is presentlypreferred to practice. It will be understood that this example isillustrative, and that the invention is not to be considered asrestricted thereto except as indicated in the appended claims.

EXAMPLE Cloning of FnuDI Restriction Endonuclease Gene

1. DNA purification: 10 g of frozen Fusobacterium nucleatum D cells werethawed on ice for 1 hour then resuspended in 20 ml of 25% sucrose, 50mMTris pH 8.0. 10 ml of 0.25M EDTA pH 8.0, and 6 ml of 10 mg/ml lysozymein 0.25M Tris pH 8.0 were added. The suspension was kept on ice for 2hours, then lysed by the addition of 24 ml of 1% Triton X-100, 50 mMTris pH 8.0, 67 mM EDTA and 5 ml of 10% SDS. The solution was extractedwith 70 ml of phenol, (previously equilibrated with 0.5M Tris pH 8.0),and 60 ml of Chloroform. The emulsion was centrifuged at 10K rpm for 30minutes to separate the phases. The viscous upper phase was transferredto a new bottle and extracted with phenol and chloroform once more. Theemulsion was again centrifuged then the upper phase was dialyzed againstfour changes of DNA buffer (10 mM Tris pH 8.0, 1 mM EDTA). The dialyzedsolution was then digested with RNase at a final concentration of 200ug/ml for 1 hour at 37° C. The DNA was then precipitated by the additionof 5M NaCl to a final concentration of 0.4M, and 0.55 volumes ofisopropyl alcohol. The precipitated DNA was spooled onto a glass rod,air-dried, then dissolved in DNA buffer to a concentration ofapproximately 300 ug/ml and stored at 4° C.

2. Digestion of DNA: 30 ug of F.nucleatum D DNA was diluted into 300 ulof restriction endonuclease digestion buffer (10mM Tris pH 7.5, 10 mMMgCl₂, 10 mM mercaptoethanol, 50 mM NaCl). 60 units of HindIIIrestriction endonuclease were added and the solution was incubated at37° C. for 2 hr. Digestion was terminated by heating to 72° C. for 12minutes.

3. Ligation and transformation: 6 ug (60 ul) of HindIII-digestedF.nucleatum D DNA was mixed with 3 ug (30 ul) of HindIII-cleaved anddephosphorylated pBR322 (ATCC 37017). 20 ul of 10 X ligation buffer (500mM Tris pH 7.5, 100 mM MgCl₂, 100 mM DTT, 5 mM ATP), and 90 ul ofsterile distilled water were added to bring the volume to 200 ul. 7.5 ulof T4 DNA ligase was added and the solution was incubated at 17° C. for4 hours. The solution was sterilized by extraction with 20 ul ofchloroform, then clarified by microcentifugation for 15 sec. 100 ul ofthe ligation solution was mixed with 800 ul of SSC/CaCl₂ (50 mM NaCl, 5mM Na₃ Citrate, 67 mM CaCl₂) and 1.7 ml of ice-cold, competent E.coliRR1 (ATCC 31343) cells were added. The solution was incubated at 44° C.for 4 mins, then 10 ml of Luria-broth (L-broth) was added and incubationwas continued at 37° C. for 3 hr.

4. Cell Library: The transformed culture was gently centrifuged, thesupernatant was discarded and the cells were resuspended inapproximately 1.2 ml of L-broth. The resuspended cells were plated inapproximately 200 ul portions onto 8 Luria-agar (L-agar) platescontaining 100 ug/ml ampicillin. The plates were incubated overnight at37° C. The transformed cells that grew up on the surfaces of the plateswere collected together by flooding each of the plates with 2 5 ml of 10mM Tris pH 7.5, 10 mM MgCl₂, scraping the colonies together, and poolingthe suspensions into a single tube.

5. Plasmid Library: 2.0 ml of the cell library was inoculated into 500ml of L-broth containing 100 ug/ml ampicillin. The culture was shakenovernight at 37° C. then centrifuged at 4K rpm for 5 minutes. Thesupernatant was discarded and the cell pellet was resuspended in 10 mlof 25% sucrose, 50 mM Tris pH 8.0, at room temperature. 5 ml of 0.25MEDTA, pH 8.0, and 3 ml of 10 mg/ml lysozyme in 0.25M Tris pH 8.0 wereadded. The solution was kept on ice for 1 hour, then 12 ml of 1% TritonX-100, 50 mM Tris pH 8.0, 67 mM EDTA was added and the suspension wasgently swirled to induce cell lysis.

The lysed mixture was transferred to a 50 ml tube and centrifuged for 45min. at 17K rpm, 4° C. The supernatant was removed with a pipette. 20.0gm of solid CsCl was weighed into a 50 ml plastic screw-cap tube and22.0 gm of supernatant was pipetted into the tube and mixed. 1.0 ml of 5mg/ml ethidium bromide in 10 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA wasadded. The solution was transferred to two 5/8 in.×3 in. centrifugetubes and spun in a Beckman Ti70 rotor for 42 hours at 44K rpm, 17° C.To collect the plasmids, the tubes were opened, illuminated withultraviolet light, and the lower of the two fluorescent bands wascollected by syringe. The lower band from each tube was combined and theethidium bromide was removed by extracting four times with an equalvolume of water-saturated, ice-cold N-Butanol.

The extracted solution was dialyzed against 4 changes of DNA buffer,then the nucleic acid was precipitated by the addition of 2 vols. ofisopropanol and sufficient 5M NaCl to reach a final concentration of0.4M. The solution was stored overnight at -20° C. then centrifuged for15 min. at 15K rpm, 0° C. The supernatant was discarded, the pellet wasair-dried for 15 min. then dissolved in 500 ul of 10 mM Tris pH 7.5, 1mM EDTA and stored at -20° C. The plasmid DNA concentration was found tobe approximately 100 ug/ml.

6. Digestion of the Plasmid Library: 6 ug (60 ul) of the plasmid librarywas diluted into 450 ul of restriction endonuclease digestion buffer(section 2). 60 units (7.5 ul) of HaeIII restriction endonuclease wereadded and the tube was incubated at 37° C. for 1 hr. The reaction wasterminated by heating to 72° C. for 12 minutes.

7. Transformation: 12.5 ul (0.18 ug) aliquots of the digested librarywere mixed with 100 ul of SSC/CaCl₂ (section 3) and 200 ul of ice-cold,competent, E.coli RR1. The mixtures were warmed to 42° C. for 3 min.then plated onto L-agar plates containing 100 ug/ml ampicillin. Theplates was incubated overnight at 37° C. HaeIII digestion was found toreduce the number of transformants by a factor of approximately 10³.Fourteen colonies were picked from the survivors of the HaeIIIdigestion; each was inoculated into 10 ml of L-broth containingampicillin, to prepare a miniculture, and streaked onto an L-agar platecontaining ampicillin, to prepare a master stock.

8. Analysis of surviving individuals: Fourteen of the surviving coloniesdescribed in section 7 were grown into 10 ml cultures and the plasmidsthat they carried were prepared by the following miniprep purificationprocedure, adapted from the method of Birnboin and Doly, Nucleic AcidsRes. 7: 1513 (1979).

Miniprep Procedure: Each culture was centrifuged at 8K rpm for 5minutes; the supernatant was discarded and the cell pellet wasresuspended in 1.0 ml of 25 mM Tris, 10 mM EDTA, 50 mM glucose, pH 8.0,containing 1 mg/ml lysozyme. After 10 minutes at room temperature, 2.0ml of 0.2M NaOH, 1% SDS was added to each tube and the tubes were shakento lyse the cells, then placed on ice. Once the solutions had cleared,1.5 ml of 3M sodium acetate, pH 4.8, was added to each and shaken. Theprecipitates that formed were spun down at 15K rpm, 4° C. for 10minutes. Each supernatant was poured into a centrifuge tube containing 3ml of isopropanol and mixed. After 10 minutes at room temperature, thetubes were spun at 15K rpm for 10 minutes to pellet the precipitatednucleic acids. The supernatants were discarded and the pellets wereair-dried at room temperature for 30 minutes. Once dry, the pellets wereresuspended in 850 ul of 10 mM Tris, 1 mM EDTA, pH 8.0. 75 ul of 5M NaClwas added to each and the solutions were transferred to Eppendorf tubescontaining 575 ul of isopropanol, and again precipitated for 10 minutesat room temperature. The tubes were then spun for 45 seconds in amicrofuge, the supernatants were discarded and the pellets wereair-dried. The pellets were then dissolved in 500 ul of 10 mM Tris, 1 mMEDTA, pH 8.0, containing 100 ug/ml RNase and incubated for 1 hour at 37°C. to digest the RNA. The DNA was precipitated once more by the additionof 50 ul of 5M NaCl followed by 350 ul of isopropanol. After 10 minutesat room temperature, the DNA was spun down by centrifugation for 45seconds, the supernatants were discarded and the pellets wereredissolved in 150 ul of 10 mM Tris 1 mM EDTA, pH 8.0. The plasmidminipreps were subsequently analyzed by digestion with FnuDI andHindIII.

9. FnuDI Methylase Gene Clones: Eight of the 14 plasmids analyzed werefound to be sensitive to HaeIII digestion and to carry diverse fragmentsof F.nucleatum D DNA. These plasmids were discarded. The remaining 6plasmids were found to be resistant to HaeIII digestion and to carry a5.5 Kb HindIII fragment in common. These plasmids, typical of which ispFnuDIRM 2-33 , appeared to be identical. Several of them were chosenand were shown to encode not only the FnuDI modification methylase butalso the FnuDI restriction endonuclease.

10. FnuDI Restriction Gene Clone: pFnuDIRM 2-33, and similar plasmids,were found to encode and express the FnuDI restriction endonuclease byassaying extracts of E.coli RR1 that carried the plasmids. 50 mlcultures of the clones were grown overnight at 37° C. in L-brothcontaining 100 ug/ml ampicillin. The cultures were centrifuged at 5K rpmfor 5 min and the cell pellets were each resuspended in 3 ml of celllysis buffer (l0 mM Tris pH 7.5, 10 mM mercaptoethanol, 0.1 1 MM EDTA).0.5 ml of 10 mg/ml lysozyme in the same buffer was added to each and thesuspensions were left on ice for 2 hr. The suspensions were frozenovernight at -20° C., then thawed on ice and 3.5 ml of cell lysis buffercontaining 0.005% Triton X-100 was vigorously mixed in to each to inducecell lysis. The lysed solutions were microcentrifuged for 5 min tocompress the nucleic acids and the supernatants were assayed forendonuclease activity in the following way:

35 ug of phage lambda DNA was diluted into 700 ul of restrictionendonuclease digestion buffer (section 2). The solution was dispensedinto 6 tubes, 150 ul into the first tube and 100 ul into each of theremaining 5 tubes. 7.5 ul of the extract was added to the first tube toachieve 1 ul extract/ug DNA. 50 ul was then removed from the first tubeand transferred to the second tube to achieve 0.3 ul/ug. 50ul serialtransfers were continued into tubes number 3 (0.1 ul/ug), 4 (0.03 ul/ug)and 5 (0.001 ul/ug). the sixth tube received no extract and served as anegative control. The tubes were incubated at 37° C. for one hour, then20 ul from each was analyzed by gel electrophoresis. The extracts werefound to contain approximately 3×10⁴ units of FnuDI restrictionendonuclease per ml, which corresponds to about 1×10⁶ units per gram ofcells (FIG. 3).

11. Transfer of the 5.5Kb fragment to pUC19: 5 ug (50 ul) of purifiedpFnuDIRM 2-33 DNA was prepared in 100 ul of restriction endonucleasedigestion buffer (section 2) containing 20 units of HindIII restrictionendonuclease. The solution was incubated at 37° C. for 1 hr. thendigestion was halted by heating at 72° C. for 12 min. 3 ug (60 ul) ofthe digested pFnuDIRM 2-33 DNA was mixed with 1.5 ug (7.5 ul) ofHindIII-cleaved and dephosphorylated pUC19 (ATCC 37254). 10 ul of 10 Xligation buffer (section 3) and 22.5 ul of sterile distilled water wereadded to bring the volume to 100 ul. 4 ul of T4 DNA ligase was added andthe solution was incubated at 17° C. for 4 hr. The ligation wassterilized by extraction with 15 ul of chloroform, then clarified bybrief microcentrifugation.

12.5 ul of the sterile ligation was mixed with 100 ul CaCl₂ /SSC(section 3) and 200 ul of competent, ice-cold, E.coli RR1. The mixturewas incubated at 42° C. for 3 min, then plated onto an L-agar platecontaining ampicillin. The plate was incubated overnight at 37° C. Thetransformants were collected by flooding the plate with 2.5 ml of 10 mMTris ph 7.5, 10 mM MgCl₂ and scraping the colonies together to form apool. The pool was inoculated into 500 ml of L-broth containingampicillin, and a plasmid preparation was purified (section 5). 5 ug ofthe purified plasmid was digested with 40 units of HaeIII restrictionendonuclease in 100 ul of restriction endonuclease buffer (section 6).12.5 ul of the digested DNA was transformed into E.coli RR1 (section 7)and survivors were recovered by plating onto L-agar plates containingampicillin and incubating the plates overnight at 37° C.

Fourteen transformants were picked and screened by the miniprepprocedure (section 8) to identify plasmids composed of pUC19 with the5.5 Kb fragment inserted at the HindIII site. Thirteen of the plasmidswere found to possess this structure; they carried the 5.5 Kb fragmentand they exhibited complete resistance to HaeIII digestion. Cellextracts of E.coli RR1 carrying two of these plasmids, pFnuDIRM 102-1and pFnuDIRM 102-4, were assayed for FnuDI restriction endonucleaseactivity. A sample of pFnuDIRM 102-1 has been deposited at the AmericanType Culture Collection under ATCC Accession No. 40521. The extractswere found to contain approximately 1×10⁵ units of FnuDI restrictionendonuclease per ml of extract, which corresponds to 3×10⁶ units/gmcells.

E.coli RR1 carrying pFnuDIRM 2-33 or pFnuDIRM 102-1 are the preferredhosts from which the FnuDI restriction endonuclease can be purified. Thestrains should be grown to stationary phase at 37° C. in a fermenter, inL-broth containing ampicillin. The cells should then be collected bycentrifugation and either broken immediately for extract preparation, orstored frozen at -70° C. until it is convenient to do so.

What is claimed as:
 1. Isolated DNA coding for the FnuDI restrictionendonuclease, wherein the isolated DNA is obtainable from the vectorpFnuDIRM 102-1.
 2. A recombinant DNA vector comprising a vector intowhich a DNA segment coding for the FnuDI endonuclease produced byFusobacterium nucleatum D has been inserted.
 3. Isolated DNA coding forthe FnuDI restriction endonuclease and methylase, wherein the isolatedDNA is obtainable from the vector pFnuDIRM 102-1.
 4. A cloning vectorwhich comprises the isolated DNA of claim
 1. 5. A cloning vector whichcomprises the isolated DNA of claim
 3. 6. The cloning vector of claim 5,wherein the cloning vector comprises pFnuDIRM 102-1.
 7. A host celltransformed by the vector of claim 4, 5 or
 6. 8. A method of producingFnuDI restriction endonuclease comprising culturing a host celltransformed with the vector of claim 4, 5 or 6 under conditions suitablefor the expression of said endonuclease.